脲和盐酸胍诱导过氧化氢酶去折叠的研究

用荧光相图法分别研究了脲和盐酸胍诱导牛肝过氧化氢酶去折叠的过程。当脲 浓度从0依次增大至0.50,4.5和8.0 mol/L时,过氧化氢酶从天然四聚体依次转变 为蓬松的四聚体、部分折叠的无活性二聚体和去折叠态,而当盐酸胍浓度从0依次 变化至0.65,2.5和6.0 mol/L时,过氧化氢酶则从天然四聚体集资转变为部分折叠 的激活二聚体、部分折叠的单体和去折叠态,这表明无论是用脲还是用盐酸胍作为 变性剂,该蛋白的变性过程都符合“四态模型”,但这两种变性剂诱导该蛋白去折 叠的途径和机制有较大差异。实验结果表明荧光相图法可以检测蛋白质去折叠的中 间态。用等温滴定量去热法研究了盐酸胍诱导过氧化氢酶去折叠过程的热力学, 25.0 ℃时低浓度盐酸胍诱导该蛋白从天然四聚体转变为部分折叠的激活二聚体的 本征摩尔构象变化焓、Gibbs自由能和熵分别为-69.2 kJ·mol~(-1),6.43 kJ· mol~(-1)和-254 J·K~(-1)·mol~(-1),据此推断盐酸胍通过熵效应和静电效应来 稳定和激活该二聚体。

Studies on the Unfolding of Catalase Induced by Urea and Guanidine Hydrochloride

The unfolding of bovine liver catalase induced by urea or guanidine hydrochloride (GuHCl) has been studied by "phase diagram" method of fluorescence. With increasing the concentration of urea from 0 to 0.50, 4.5 and 8.0 raol/L, the conformation of catalase is changed from the native tetramer to an expanded tetramer, a partially folded inactive dimer and the unfolded state, respectively. With increasing the concentration of GuHCl from 0 to 0.65, 2.5 and 6.0 raol/L, however, the conformation of catalase is changed from the native tetramer to a partially folded activated dimer, a partially folded monomer and the unfolded state, respectively. This indicates that the unfolding of catalase induced by urea or GuHCl follows a four-state model, but both the pathway and the mechanism for the unfolding of catalase induced by urea are different from those induced by GuHCl. The experimental results show that the "phase diagram" method of fluorescence can be used to detect unfolding intermediates of proteins. The unfolding of catalase induced by GuHCl has been studied by isothen-nal titration calorimetiy. The intrinsic enthalpy, Gibbs free energy and entropy changes for formation of the partially folded activated dimer of the protein in the presence of low GuHCl concentrations at 25.0 ℃ were measured as - 69.2 kJ·mol~(-1), 6.43 kj·mol~(-1) and - 254 J· K~(-1) ·mol~(-1) respectively. It is thus indicated that the stabilization and activation of this partially folded dimer by GuHCl originate from the entropic and the electrostatic effect of this denaturant.